Chemistry Reference
In-Depth Information
light is injected from both sides of the glass slide, so that the generated evanescent
wave is emitted from the glass surface to an extremely restricted space, within
approximately 200 nm. As a result, only the bound fl uorescent molecules are sub-
stantially excited by this near- optic - fi eld excitation principle. Good signal-to-noise
ratios are obtained, without washing, in a manner preserving binding equilibrium.
Assuming that the above principle is valid, it should be possible to detect weak
interactions that other methods cannot reveal.
To test this method, tetramethylrhodamine ( TMR ) - labeled biantennary galac-
tosylated
N
-glycan (generally called NA2) was applied to the lectin microarray (for
N
-glycan structures, please see Chapters 6 and 8) [16]. After incubation, the glass
slide was directly measured by the evanescent-type scanner. The fl uorescent
signal was specifi cally observed on
Ricinus communis
agglutinin ( RCA120 ) (see
the fi rst image on the left in Figure 14.5b), which is known to recognize the
Lac/LacNAc unit. The RCA120 intensity was preserved even when the scan was
made later (the second image from the left in Figure 14.5b). On the other hand,
if the incubation bath (well on the glass slide) was rinsed with buffer, the detect-
able signals dramatically decreased in intensity (the third image from the left in
Figure 14.5b). This observation unambiguously shows that the developed system
has the great advantage of being able to detect relatively weak interactions in an
equilibrium state.
14.6
Practice in Differential Glycan Profi ling: Approaches and Applications
To observe lectin-glycan interactions in terms of fl uorescent signals on the lectin
microarray, two approaches to fl uorescence labeling of glycoconjugates can be
used, direct and indirect methods. In the direct-labeling method, appropriate fl uo-
rescent reagents (e.g., Cy3-succinimidyl ester) are used to label target glycoconju-
gates, directly prior to a binding reaction. For this purpose, various chemical
cross-linkers (e.g., succimidyl ester and maleimide) are commercially available,
which enable profi ling of a mixture of glycoproteins from whole-cell lysates. It is
a general consensus of glycobiologists that each cell type (origin of species and
tissue) and state (differential stage and malignancy) directs different expression
of glycoconjugates on cells. In other words, each cell is defi ned by its own glycome.
Based on this concept of differential glycomics, the lectin microarray approach can
be used effi ciently to discriminate complex and heterogeneous features of glycans
expressed on cells [14, 15]. As an application of lectin microarray, a novel proce-
dure was developed for direct cell analysis, in which the fl uorophore Cell - Tracker
Orange CMRA is incorporated and metabolized to generate a fl uorescent com-
pound (Figure 14.6) [17]. This method enables direct profi ling of cell-surface
glycans. The developed methodology should also contribute to the elucidation of
physiological functions of endogenous animal lectins in the context of ' real gly-
comics' (please see also Table 25.1 and [18] for design of glycan arrays to analyze
lectin specifi city).
